Mutated alkaline cellulase

ABSTRACT

A mutated alkaline cellulase derived from a cellulase having the amino acid sequence represented by SEQ ID NO: 1 or one having a homology of at least 90% therewith by deleting one or more amino acid residues from the 343- to 377-positions in SEQ ID NO: 1 or a region corresponding thereto and then inserting a peptide having from 2 to 15 amino acid residues into the deletion site; and a gene encoding the same. The above alkaline cellulase has an optimum pH value close to the pH value of laundry water and, therefore, is useful as an enzyme for detergents.

TECHNICAL FIELD

The present invention relates to mutated alkaline cellulases which canbe incorporated in laundry detergents or the like.

BACKGROUND ART

When a laundry detergent is used for washing laundry, the pH of thewashing liquid is mostly from 10 to 11; i.e., within an alkaline range.Therefore, an enzyme to be incorporated into laundry detergents isrequired to exhibit an optimum pH in an alkaline region and to be stableunder an alkaline condition.

Conventionally known alkaline cellulases which can be incorporated intolaundry detergents or other detergents include alkaline cellulasederived from Bacillus sp. KSM-635 belonging to Bacillus (Japanese PatentPublication (kokoku) No. 60-23158, Japanese Patent Publication (kokoku)No. 6-030578, U.S. Pat. No. 4,945,053, etc.); alkaline cellulase derivedfrom Bacillus sp. KSM-64 (Shikata et al. Agric. Biol. Chem., 54, 91-96,1990, Sumitomo et al., Biosci. Biotechnol. Biochem., 56, 872-877, 1992);heat-resistant alkaline cellulase produced from mesophilic andalkaliphilic fungi, Bacillus sp. KSM-S237 (FERM-BP7875: deposited onFeb. 6, 1997 with Independent Administrative Institution of NationalInstitute of Advanced Industrial Science and Technology, InternationalPatent Organism Depositary (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba,Ibaraki, Japan (postal code 305-8566))) (Japanese Patent ApplicationLaid-Open (kokai) No. 10-313859); alkaline cellulase derived fromBacillus sp. KSM-N257 (Japanese Patent Application No. 12-281378); andalkaline cellulase derived from Bacillus sp. KSM-N131 (Japanese PatentApplication No. 12-373859). However, in the case where the substrate iscarboxymethylcellulose (CMC) these alkaline cellulases exhibit anoptimum reaction pH of about 9; therefore, the cellulases do not havethe optimum pH under conditions encountered during laundry washing.

In the meanwhile, a study has been done on changing the optimum reactionpH of a glucosidase. The study shows that the optimum pH of aglucosidase is shifted from alkali to neutral by constructing a chimericprotein from an alkaline (NK1) cellulase derived from alkaliphilicBacillus and neutral cellulase (BSC) derived from Bacillus subtilis(Park et al. Protein Enz. 6, 921-926, 1993).

Recently, it has been reported that the optimum pH of cellobiohydrolase(Cel7A) derived from Trichoderma reesei was increased as compared withthat of its wild-type strain by substitution of amino acid residues inthe vicinity of its active center (Beker et al, Biochem. J., 356, 19-31,2001). However, in this case, the optimum pH of the wild-type enzymefalls within an acidic range, and the increase in the optimum pH of themutant is within 1 pH unit or less.

Thus, there has been substantially no report in which the optimumreaction pH of glucosidase is shifted toward the alkaline side.

The present invention is directed to the provision of a mutated alkalinecellulase having an optimum pH as an enzyme to be incorporated intodetergent, which is obtained by modifying the alkaline cellulase gene.

DISCLOSURE OF THE INVENTION

The present inventors have searched enzymes which can attain the abovepurpose by estimating the three-dimensional structure of an alkalinecellulase having an amino acid sequence represented by SEQ ID NO: 2(Egl-237); in particular, the structure of its active domain, and byincorporating mutations through site-specific mutation. As a result, thepresent inventors have found that the optimum reaction pH in the CMCdecomposition activity can be increased by deleting amino acid residuesin a specific region which forms a portion of the loop structure andinserting a peptide into the position.

Accordingly, the present invention provides a mutated alkaline cellulaseobtained by deleting, from a cellulase having an amino acid sequencerepresented by SEQ ID NO: 2 or an amino acid sequence exhibiting atleast 90% homology therewith, one or more amino acid residues chosenfrom the 343rd to 377th positions in SEQ ID NO: 2 or from correspondingpositions, and inserting a peptide having 2 to 15 amino acid residuesinto at least one of the deleted positions; as well as a gene encodingthe mutated alkaline cellulase.

The present invention also provides a vector containing the gene, and atransformant containing the vector.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a to 1 c show aligned amino acid sequences of cellulases havingat least 90% homology with the amino acid sequence represented by SEQ IDNO: 2 (Egl-237). Egl-1139 appears as SEQ ID NO: 7, Egl-64 appears as SEQID NO: 8, and Egl-N131b appears as SEQ ID NO: 9.

FIG. 2 shows the optimum reaction pH of the alkaline cellulase which hasbeen mutated through use of alanine-glycine-alanine.

FIG. 3 shows the optimum reaction pH of the alkaline cellulase which hasbeen mutated through use of alanine-histidine-alanine.

FIG. 4 shows the optimum reaction pH of the alkaline cellulase which hasbeen mutated through use of alanine-arginine-alanine.

BEST MODE FOR CARRYING OUT THE INVENTION

The mutated alkaline cellulases according to the present invention areobtained by using, as a cellulase to be mutated (hereinafter may bereferred to as “parent alkaline cellulase”), a cellulase having an aminoacid sequence represented by in SEQ ID NO: 2 or an amino acid sequenceexhibiting at least 90% homology therewith, and by deleting one or moreamino acid residues chosen from the 343rd to 377th positions in SEQ IDNO: 2 or from corresponding positions and inserting a peptide having 2to 15 amino acid residues into at least one of the deleted positions.The parent alkaline cellulases may be obtained either throughspontaneous or artificial mutation of the cellulase having the aminoacid sequence of SEQ ID NO: 2.

The parent cellulase exhibiting 90% or more homology with the amino acidsequence represented by SEQ ID NO: 2 preferably exhibits 95% or morehomology, more preferably 98% or more homology, with the amino acidsequence. The cellulase may be of wild-type or a mutant of a wild-typecellulase. The homology of an amino acid sequence can be calculated bymeans of a program such as maximum matching or search homology ofGENETYX-WIN (Software Development Co.).

When the molecular structure of the cellulase exhibiting 90% or morehomology with the amino acid sequence represented by SEQ ID NO: 2 isestimated through a homology modeling technique and by means of 3D-1D,XPLORE, and PROCHECK programs, the cellulase preferably has thefollowing two characteristics; (i) the cellulase has an amino acidsequence exhibiting 70% or more homology, more preferably 80% or morehomology, much more preferably 90% or more homology, still morepreferably 95% or more homology, yet still more preferably 98% or morehomology, with the region from the 42nd position to the 404th position(valine) (i.e., the active domain region) (i.e., the active domainregion) of SEQ ID NO: 2; and (ii) the region from the 343rd position(asparagine) to the 377th position (leucine) of SEQ ID NO: 2 has a loopstructure in the cellulase molecule. The homology of an amino acidsequence may be calculated in accordance with, for example, theLipman-Pearson method (Science, 227, 1435, 1985).

In addition, the parent alkaline cellulase preferably hascharacteristics such as the followings: having a molecular weight of86,000±2,000 as measured through sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) or gel filtration; having an optimumreaction pH of from 7.5 to 9.0 in the case where the substrate iscarboxymethylcellulose; and having an optimum reaction temperaturefalling within a range of from 40 to 50° C. In addition, it ispreferable that the parent alkaline cellulase effectively digestslichenan as well as carboxymethylcellulose and sufficiently maintains astability when treated at pH 9 and at 50° C. for 10 minutes.

More preferably, the parent alkaline cellulase has the followingcharacteristics: having a molecular weight of 86,000±2,000 (as measuredthrough SDS-PAGE or gel filtration employing a Sephacryl S200 column);having an optimum reaction pH of from 8.6 to 9.0 and an optimum reactiontemperature of 50° C.; effectively digesting lichenan as well ascarboxymethylcellulose; exhibiting a remaining activity of 95% or more,where the remaining activity after treatment at 30° C. for 10 minutes istaken as 100%, after being treated at pH 9 and 50° C. for 10 minutes inthe presence of 5 mM calcium chloride.

Accordingly, the parent alkaline cellulase of the present invention ispreferably, in addition to the alkaline cellulase having the amino acidsequence represented by SEQ ID NO: 2, an alkaline cellulase having (i)the above amino acid sequence features-i.e., having a high homology inthe active domain region of SEQ ID NO: 2 and containing a particularregion having a loop structure in the cellulase molecule as describedabove-and/or the above enzymatic characteristics (particularlypreferably, having the amino acid sequence features and the enzymaticcharacteristics in combination), and (ii) an amino acid sequenceexhibiting 90% or more homology (preferably 95% or more homology, muchmore preferably 98% or more homology) with that represented by SEQ IDNO: 2.

Examples of the parent alkaline cellulase of the present inventioninclude Egl-237 [derived from Bacillus sp. KSM-S237 (FERM BP-7875),which is “alkaline cellulase having the amino acid sequence representedby SEQ ID NO: 2,” Hakamada et al., Biosci. Biotechnol. Biochem., 64,2281-2289, 2000]; alkaline cellulases derived from Bacillus sp. strain1139 (Egl-1139; SEQ ID NO: 7) (Fukumori et al., J. Gen. Microbiol., 132,2329-2335) (homology: 91.4%); alkaline cellulases derived from Bacillussp. strain KSM-64 (Egl-64; SEQ NO: 8) (Sumitomo et al., Biosci.Biotechnol. Biochem., 56, 872-877, 1992) (homology: 91.9%); andcellulases derived from Bacillus sp. strain KSM-N131 (Egl-N131b; SEQ IDNO: 9) (Japanese Patent Application No. 2000-47237) (homology: 95.0%).

The mutated alkaline cellulase of the present invention is obtained bydeleting, from the parent alkaline cellulase, one or more amino acidresidues chosen from the 343rd to 377th positions in SEQ ID NO: 2 orfrom corresponding positions and inserting a peptide having 2 to 15amino acid residues into at least one of the deleted positions.

The amino acid residue(s) to be deleted may be any of 35 amino acidresidues included in the 343rd to 377th positions of SEQ ID NO: 2. Thenumber of the amino acid residue(s) to be deleted may be any of 1 to 35.The amino acid residues to be deleted are continuous or non-continuous.The amino acid residue(s) to be deleted is(are) preferably included inthe 350th to 377th positions, more preferably in the 355th to 365thpositions, much more preferably in the 357th to 362nd positions, of SEQID NO: 2.

More preferably, the amino acid residue(s) to be deleted is(are) any 1to 27 residues, any 2 to 15 residues, or any 3 to 10 residues containedin the 343rd to 377th positions; any 1 to 8 residues, any 3 to 6residues, or all the amino acid residues contained in the 355th to 377thpositions; and any 2 residues, any 2 to 5 residues, or all the aminoacid residues contained in the 357th to 362nd positions.

Three-dimensional structural analysis through homology modeling (Ozawaei al., Protein Eng., 14, 501-504, 2001) suggests that the amino acidregion at the 343rd to 377th positions of SEQ ID NO: 2 is locatedrelatively distant from the active center of Egl-237 and therefore has ahigh degree of freedom, and is suggested to be a region that forms aportion of the loop structure that is intimately involved in maintainingthe cellulase structure.

The “amino acid residue corresponding to the 343rd to 377th positions ofSEQ ID NO: 2” can be identified by comparing amino acid sequences bymeans of a known algorithm such as Lipman-Pearson's method, and aligningthe amino acid residues contained in the amino acid sequences of thealkaline cellulases such that the homology of each amino acid sequencewith respect to that of SEQ ID NO: 2 is maximized. By aligning the aminoacid sequence of the cellulase in such a manner, the position of thehomologous amino acid residue in the amino acid sequence of eachcellulase can be determined, irrespective of insertion or deletion inthe amino acid sequence (FIG. 1). The homologous position is presumed toexist at the same three-dimensional position and to bring about similareffects with regard to a specific function of the target cellulase.

Table 1 shows the positions of Egl-1139 (SEQ ID NO: 7), Egl-64 (SEQ IDNO: 8), and Egl-N131b (SEQ ID NO: 9) corresponding to the 357th to 362ndpositions of alkaline cellulase having an amino acid sequencerepresented by SEQ ID NO: 2 (Egl-237).

TABLE 1 Egl-237 Egl-1139 Egl-64 Egl-N131b 357Gly 357Gly 357Gly 343Gly358Lys 358Lys 358Lys 344Lys 359Ser 359Ser 359Ser 345Ser 360Asn 360Asn360Asn 346Asn 361Ala 361Ala 361Ala 347Ala 362Thr 362Thr 362Thr 348Thr

The peptide to be inserted into the deleted position(s) may be formed ofany of 20 essential amino acids. The peptide preferably containsalanine, glycine, histidine, or arginine. More preferably, the peptidecontains alanine and glycine, alanine and histidine, or alanine andarginine.

The number of the amino acid residues forming the peptide to be insertedis preferably from 2 to 15, more preferably from 2 to 10, much morepreferably from 2 to 6, particularly preferably 3.

Preferred examples of the peptide to be inserted includeasparagine-threonine-alanine-valine-glycine-isoleucine,alanine-serine-methionine-leucine-phenylalanine-glutamic acid,cysteine-leucine-glycine-histidine-serine,tyrosine-glutamine-lysine-alanine-alanine, asparticacid-methionine-isoleucine-valine, isoleucine-threonine-proline-lysine,glycine-leucine-cysteine, and serine-valine-phenylalanine, inter alia, apeptide containing alanine residues at both ends thereof and having 3 to6 residues; more preferably alanine-any one amino acid-alanine; evenmore preferably alanine-glycine-alanine, alanine-histidine-alanine, oralanine-arginine-alanine.

The mutated alkaline cellulases of the present invention encompassesthose having one to several amino acid residues deleted, replaced, oradded at position(s) in the amino acid sequence other than the mutatedposition(s) described above, so far as they do not lose their alkalinecellulase activity and the modified characteristics described above.

The mutated alkaline cellulase of the present invention may be obtainedthrough incorporating a desired mutation into a parent alkalinecellulase in a manner, for example, described below.

Specifically, a parent alkaline cellulase is cultured, and the resultantculture broth is centrifuged, to thereby isolate cells. Through use ofthe alkaline cellulase gene collected from the cells, a chromosomal DNAcontaining the alkali cellulase gene is prepared [through, for example,a method of Marmar (J. Mol. Biol., 3, 208-212, 1961) or a method ofSaito and Miura (Biochim. Biophys. Acta, 72, 619-629, 1963)]. Thechromosomal DNA may be subjected to cloning through shotgun cloning orPCR, to thereby prepare a gene (SEQ ID NO: 1) encoding the parentalkaline cellulase (e.g., alkaline cellulase having an amino acidsequence represented by SEQ ID NO: 2). To the thus-obtained gene, amutation is introduced, and the resultant mutated gene is incorporatedto a plasmid. Appropriate host cells are transformed through use of theplasmid and then cultured, and the mutated alkaline cellulase of thepresent invention may be collected from the culture.

Examples of the method which may be employed to introduce a mutationinto a gene encoding a parent alkaline cellulase, include site-specificmutation. For example, a Site-Directed Mutagenesis System Mutan-SuperExpress Km kit (product of Takara) may be used. The mutated alkalinecellulase gene may be incorporated into an appropriate vector.

The vector may be any vector which satisfies the following threeconditions: (i) the vector can be replicated and maintained in hostcells; (ii) the vector allows expression of the alkaline cellulase gene;and (iii) the vector can stably maintain the gene incorporated therein.When the host cell is a bacterium belonging to Bacillus, examples of thevector to be used include pUB110 and pHY300PLK. When the host cell is E.coli, examples of the vector to be used include pUC18, pUC19, pBR322,and pHY300PLK.

Transformation of the host bacteria through use of the thus-obtainedrecombinant vector may be carried out through, for example, theprotoplast method, the competent cell method, or the electroporation.Examples of the host bacteria include, but are not limited to,gram-positive bacteria such as those belonging to Bacillus sp. (Bacillussubtilis), gram-negative bacteria such as Escherichia coli, fungi suchas those belonging to Streptomyces (Actinomycetes), to Saccharomyces(yeast), and to Aspergillus (molds).

The thus-obtained transformant may be cultured under proper conditionsusing a medium containing an assimilable carbon source, nitrogen source,metal salt, vitamin, etc. The enzyme is isolated from the thus-obtainedculture broth and then purified through a routine method, followed bylyophilization, spray drying, or crystallization to obtain the enzyme ina desired form.

The thus-obtained mutated alkaline cellulase has an optimum reaction pHhigher than that of the parent alkaline cellulase. The optimum reactionpH lies preferably within 9.0 to 9.5, more preferably within 9.5 to ashigh as 10.0. In addition, other than the optimum reaction pH, themutated alkaline cellulase preferably has the same characteristics asthe parent alkaline cellulase.

EXAMPLES Example 1 Modification of Loop Region of Egl-237

The molecule model of Egl-237 was constructed through homology modelingbased on the analytical data of CelK, whose crystal structure hadalready been analyzed. Details of the model structure were determined bymeans of 3D-1D, XPLORE, and PROCHECK programs. Thereafter, on the basisof the obtained data, amino acid residues contained in a portion of theloop structure (from the 357th (glycine) to 362nd (threonine)) weredeleted, and one of alanine-glycine-alanine, alanine-histidine-alanine,and alanine-arginine-alanine was inserted to the deleted position.Mutation of the loop region was performed through use of mutationintroducing primers 1, 2, and 3 (SEQ ID NOs: 3, 4, and 5), respectively,and through use of mutation introducing primer 4 (SEQ ID NO: 6) as anantisense primer. When alanine-glycine-alanine was inserted formutation, Egl-237 gene was incorporated into pHY300PLK, and the productwas employed as a template DNA. When alanine-histidine-alanine oralanine-arginine-alanine was inserted for mutation, a plasmid composedof pHY300PLK containing a gene mutated by alanine-glycine-alanine wasemployed as a template DNA. Specifically, after mixing of 0.5 μL (10 ng)of the template DNA plasmid, 20 μL (1 μM) of the mutation introducingprimer, 20 μL (1 μM) of the antisense primer, 10 μL of a ×10 PCR buffersolution, 8 μL of a 10 mM deoxynucleotide triphosphate (dNTP) mixture,0.5 μL (2.5 units) of “Pyrobest DNA polymerase” (product of Takara), and39.5 μL of deionized water, PCR was carried out by “gene amp PCR system9700” (product of Amersham-Pharmacia). The reaction conditions were asfollows; starting with thermal denaturation at 94° C. for 2 minutes;followed by 30 cycles of 94° C. for 1 minute; 60° C. for 1 minute; 72°C. for 1.5 minutes; and finally 72° C. for 3 minutes. After purificationof the resultant PCR product by “GFX PCR DNA and Gel Band PurificationKit” (Amersham-Pharmacia) (43.5 μL), 5.5 μL of a ×10 phosphorylationbuffer and 1 μL (10 units) of polynucleotide kinase were added to thesolution, and it was maintained at 37° C. for 1 hour forphosphorylation, followed by purification. After mixing 25 μL of thephosphorylated PCR product with 2 μL (20 ng) of the template plasmid, 10μL of a ×10 PCR buffer, 8 μL of a 10 mM dNTP mixture, 1 μL (5 units) of“Pyrobest DNA polymerase,” and 54 μL of deionized water, PCR wasconducted. The reaction conditions were as follows; starting withthermal denaturation at 94° C. for 2 minutes; followed by 30 cycles of94° C. for 1 minute; 58° C. for 1 minute; 72° C. for 6 minutes; andfinally 72° C. for 12 minutes. The resultant PCR product was purified(43.5 μL). Then, 5.5 μL of a ×10 phosphorylation buffer and 1 μL (10units) of polynucleotide kinase were added thereto, and phosphorylationwas conducted at 37° C. for 1 hour. The mixture was subjected to ethanolprecipitation. The thus-collected DNA solution (10 μL) was subjected toligation at 16° C. for 18 hours by use of a ligation kit ver. 2 (productof Takara) for self ring closure, followed by another round of ethanolprecipitation, whereby the DNA mixture was collected.

Example 2 Method for Transformation

By use of 5 μL of the DNA mixture obtained in Example 1, the DNA wasintroduced into the Bacillus subtilis strain ISW1214, whereby thecorresponding transformant was obtained (Chang and Cohen, Mol. Gen.Gent., 168, 111, 1979). The thus-obtained protoplast was inoculated ontoa DM3 regeneration agar medium [0.8% (w/v) agar (product of Wako PureChemicals), 0.3M disodium succinate 6 hydrate, 0.5% “Casamino AcidsTechnical” (product of Difco), 0.5% yeast extract, 0.35% KH₂PO₄, 0.15%K₂HPO₄, 0.5% glucose, 0.4% MgCl₂.6H₂O, 0.01% bovine serum albumin(product of Sigma), 0.5% CMC (Kanto Chemical Co., Inc.), 0.005% trypanblue (product of Merck), and an amino acid mixture (leucine andmethionine, 10 μg/mL)] containing tetracycline (15 μg/mL, Sigma), andincubated at 30° C. for 72 hours to obtain a transformant. Thetransformant that formd a halo on the DM3 regeneration agar plate wascultured while shaking at 30° C. for 15 hours in a polypeptone medium(3% polypeptone S, 3% maltose, 0.5% fish meat extract (product of WakoPure Chemicals), 0.1% yeast extract, 0.1% KH₂PO₄, and 0.02% MgSO₄.7H₂O)containing tetracycline (15 μg/mL). After collection of the cells,plasmids were extracted and purified by “Micro Prep Plasmid Purificationkit” (product of Amersham-Pharmacia). The mutated nucleotide sequence ofthe cellulase gene, which had been inserted into the plasmids, wasconfirmed by means of “377DNA Sequencer” (product of AppliedBiosystems). The nucleotide sequencing was performed through use ofprimers which were suitable for determining nucleotide sequences in thevicinity of the mutated position, and screening was performed, wherebyplasmids to which the target mutation had been incorporated wereobtained.

Example 3 Production of Cellulase Mutant

The host bacteria B. Subtilis strain ISW1214 was cultured at 30° C. for72 hours in a medium containing 3% polypepton S (product of NihonPharmaceutical), 0.5% fish meat extract, 0.05% yeast extract, 0.1%KH₂PO₄, 0.02% MgSO₄.7H₂O, tetracycline (15 μg/mL), and 5% maltose.

After completion of culturing of various cellulase mutants, thecellulose activity of variants having a loop structure mutated byalanine-glycine-alanine, alanine-histidine-alanine, andalanine-arginine-alanine were found as 36800 U/L, 34700 U/L, and 32400U/L, respectively.

The cellulase activity was determined through the 3,5-dinitrosalicylicacid (DNS) method.

That is, to a reaction mixture composed of 0.2 mL of a 0.5Mglycine-sodium hydroxide buffer (pH 9.0), 0.4 mL of 2.5% (w/v)carboxymethyl cellulose (A01MC: Nippon Paper Industries), and 0.3 mL ofdeionized water, 0.1 mL of a properly diluted enzyme solution was added.After the resultant mixture was allowed to undergo reaction at 40° C.for 20 minutes, 1 mL of a dinitrosalicylic acid reagent (0.5%dinitrosalicylic acid, 30% Rochelle Salt, 1.6% aqueous sodium hydroxide)was added, and the color of a reducing sugar was developed in boilingwater for 5 minutes. After quenching in ice water, 4 mL of deionizedwater was added, and absorbance at 535 nm was measured to determine theproduction amount of the reducing sugar. A blank sample was prepared asfollows; dinitrosalicylic acid reagent was added to the reaction mixturethat had been treated as described except the addition of the enzymesolution. Then the enzyme solution was added thereto, and the color wasdeveloped in the same way. One unit (1 U) of an enzyme activity wasdefined as an amount of enzyme which produces a reducing sugar in anamount equivalent to 1 μmol of glucose in 1 minute under theabove-described reaction conditions.

Example 4 Purification of Recombinant Loop-modified Cellulase

The supernatant of the culture broth containing the recombinantloop-modified cellulase was diluted 10-fold with deionized water, andthe diluted supernatant was incorporated into a column (2.5 cm×5 cm)containing DEAE Toyopearl (Tosoh Corporation) that had beenpre-equilibrated with 10 mM Tris-HCl buffer (pH 8.0). The column waswashed further with the buffer, and protein was eluted with a lineargradient of from 0 to 0.4M solution of sodium chloride solution (400 mL)in the same buffer. The recombinant loop-modified cellulase of interestwas eluted at a sodium chloride solution concentration of approximately0.25 M and the eluted component was found substantially homogenous asanalyzed by electrophoresis. Desalting and condensation were performedthrough use of an ultrafilter (PM10, Millipore).

Example 5 Optimum Reaction pH of Recombinant Loop-modified Cellulase

The purified product of the recombinant loop-modified cellulase preparedin Example 4 was investigated in terms of the effect of pH on enzymaticreaction. Optimum reaction pH was determined by use of glycine-sodiumhydroxide buffer (pH 8.2 to 10.9). As a result, a recombinant wild-typecellulase was found to have an optimum reaction pH of 9.0, whereas thecellulase which had been modified by alanine-glycine-alanine was foundto have an optimum reaction pH of 10, which is 1 pH unit higher thanthat of the recombinant wild-type cellulase (FIG. 2); i.e., the optimumpH shifted toward higher alkaline side. The cellulase, which had beenmodified by alanine-histidine-alanine or alanine-arginine-alanine wasfound to have an optimum reaction pH of about 9.6. In addition, within arange of pH 8.8 to pH 9.9, the cellulase which had been modified byalanine-histidine-alanine or alanine-arginine-alanine was found to havea relative activity (i.e., a percent activity when the activity atoptimum reaction pH, in this case, pH 9.6, is taken as 100%) of 95% ormore, which is higher than those of the parent cellulase and thecellulase mutated by alanine-glycine-alanine (FIGS. 2 and 3).

INDUSTRIAL APPLICABILITY

The mutated alkaline cellulases of the present invention have an optimumpH near the pH of the washing liquid (pH of about 10.5), and thus areuseful as enzymes for detergents.

1. A mutated alkaline cellulase which is obtained by deleting, from acellulase having the amino acid sequence of SEQ ID NO: 2 or a homologousamino acid sequence exhibiting at least 95% sequence homology therewith,a peptide consisting of one or more amino acid residues chosen from the357^(th) to 362^(nd) positions in SEQ ID NO: 2 or from correspondingpositions of said homologous amino acid sequence and replacing thepeptide with an insertion peptide selected from the group consisting of:asparagine-threonine-alanine-valine-glycine-isoleucine,alanine-serine-methionine-leucine-phenylalanine-glutamic acid,cysteine-leucine-glycine-histidine-serine,tyrosine-glutamine-lysine-alanine-alanine, asparticacid-methionine-isoleucine-valine, isoleucine-threonine-proline-lysine,glycine-leucine-cysteine, serine-valine-phenylalanine, and a peptidecontaining alanine residues at both ends thereof and having 3 to 6residues, wherein said mutated alkaline cellulase has alkaline cellulaseactivity.
 2. The mutated alkaline cellulase as described in claim 1,wherein said insertion peptide containing alanine residues at both endsthereof and having 3 to 6 residues, contains as structural amino acidresidues thereof, alanine and glycine, alanine and histidine, or alanineand arginine.
 3. The mutated alkaline cellulase as described in claim 1,wherein said insertion peptide is selected from the group consisting ofalanine-glycine-alanine, alanine-histidine-alanine, andalanine-arginine-alanine.
 4. An isolated polynucleotide encoding amutated alkaline cellulase as recited in claim
 1. 5. A recombinantvector comprising the polynucleotide as recited in claim
 4. 6. Anisolated transformed microorganism comprising a recombinant vector asrecited in claim
 5. 7. A method for producing a mutated alkalinecellulase, which comprises culturing the isolated transformedmicroorganism of claim 6 in a medium for a time and under conditionssuitable to produce and accumulate said mutated alkaline cellulase, andisolating said mutated alkaline cellulase.
 8. The mutated alkalinecellulase as described in claim 1, wherein the homologous amino acidsequence exhibits at least 98% sequence homology to the amino acidsequence of SEQ ID NO:
 2. 9. A mutated alkaline cellulase which isobtained by deleting, from a cellulase selected from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, a peptideconsisting of one or more amino acid residues chosen from the positionscorresponding to the 357^(th) to 362^(nd) positions of SEQ ID NO: 2, andreplacing the peptide with an insertion peptide having 2 to 6 amino acidresidues, wherein said mutated alkaline cellulase has alkaline cellulaseactivity.
 10. The mutated alkaline cellulase according to claim 9,wherein said insertion peptide has 2 to 5 amino acid residues.
 11. Themutated alkaline cellulase as described in claim 9, wherein saidinsertion peptide has 3 amino acid residues.
 12. The mutated alkalinecellulase as described in claim 9, wherein said insertion peptidecontains as structural amino acid residues thereof, alanine and glycine,alanine and histidine, or alanine and arginine.
 13. The mutated alkalinecellulase as described in claim 9, wherein said insertion peptide isselected from the group consisting of alanine-glycine-alanine,alanine-histidine-alanine, and alanine-arginine-alanine.
 14. An isolatedpolynucleotide encoding a mutated alkaline cellulase as recited in claim9.
 15. A recombinant vector comprising the polynucleotide as recited inclaim
 14. 16. An isolated transformed microorganism comprising arecombinant vector as recited in claim
 15. 17. A method for producing amutated alkaline cellulase, which comprises culturing the isolatedtransformed microorganism of claim 16 in a medium for a time and underconditions suitable to produce and accumulate said mutated alkalinecellulase, and isolating said mutated alkaline cellulase.
 18. Themutated alkaline cellulase as described in claim 9, wherein saidinsertion peptide is selected from the group consisting of:asparagine-threonine-alanine-valine-glycine-isoleucine,alanine-serine-methionine-leucine-phenylalanine-glutamic acid,cysteine-leucine-glycine-histidine-serine,tyrosine-glutamine-lysine-alanine-alanine, asparticacid-methionine-isoleucine-valine, isoleucine-threonine-proline-lysine,glycine-leucine-cysteine, and serine-valine-phenylalanine.
 19. A methodof producing a mutated alkaline cellulase comprising deleting from acellulase having the amino acid of sequence of SEQ ID NO: 2 or ahomologous amino acid sequence exhibiting at least 95% sequence homologytherewith, a peptide consisting of one or more amino acid residueschosen from the 357^(th) to 362^(nd) positions in SEQ ID NO: 2 or fromcorresponding positions of said homologous amino acid sequence andreplacing the peptide with an insertion peptide having 2 to 6 amino acidresidues, wherein said mutated alkaline cellulase has alkaline cellulaseactivity.
 20. The method of claim 19, wherein said insertion peptide isselected from the group consisting of:asparagine-threonine-alanine-valine-glycine-isoleucine,alanine-serine-methionine-leucine-phenylalanine-glutamic acid,cysteine-leucine-glycine-histidine-serine,tyrosine-glutamine-lysine-alanine-alanine, asparticacid-methionine-isoleucine-valine, isoleucine-threonine-proline-lysine,glycine-leucine-cysteine, and serine-valine-phenylalanine.
 21. Themethod of claim 19, wherein said insertion peptide has 2 to 5 amino acidresidues.
 22. The method of claim 19, wherein said insertion peptide has3 amino acid residues.
 23. The method of claim 19, wherein saidinsertion peptide contains alanine residues at both ends thereof and has3 to 6 residues.
 24. The method of claim 19, wherein said insertionpeptide contains as structural amino acid residues thereof, alanine andglycine, alanine and histidine, or alanine and arginine.
 25. The methodof claim 19, wherein said insertion peptide is selected from the groupconsisting of alanine-glycine-alanine, alanine-histidine-alanine, andalanine-arginine-alanine.
 26. A method of producing a mutated alkalinecellulase comprising deleting from a cellulase selected from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, a peptideconsisting of one or more amino acid residues chosen from the positionscorresponding to the 357^(th) to 362^(nd) positions of SEQ ID NO: 2, andreplacing the peptide with an insertion peptide having 2 to 6 amino acidresidues, wherein said mutated alkaline cellulase has alkaline cellulaseactivity.
 27. The method of claim 26, wherein said insertion peptide isselected from the group consisting of:asparagine-threonine-alanine-valine-glycine-isoleucine,alanine-serine-methionine-leucine-phenylalanine-glutamic acid,cysteine-leucine-glycine-histidine-serine,tyrosine-glutamine-lysine-alanine-alanine, asparticacid-methionine-isoleucine-valine, isoleucine-threonine-proline-lysine,glycine-leucine-cysteine, and serine-valine-phenylalanine.
 28. Themethod of claim 26, wherein said insertion peptide has 2 to 5 amino acidresidues.
 29. The method of claim 26, wherein said insertion peptide has3 amino acid residues.
 30. The method of claim 26, wherein saidinsertion peptide contains alanine residues at both ends thereof and has3 to 6 residues.
 31. The method of claim 26, wherein said insertionpeptide contains as structural amino acid residues thereof, alanine andglycine, alanine and histidine, or alanine and arginine.
 32. The methodof claim 26, wherein said insertion peptide is selected from the groupconsisting of alanine-glycine-alanine, alanine-histidine-alanine, andalanine-arginine-alanine.